Novel therapeutic and diagnostic products and methods

ABSTRACT

The present invention relates to the use of p11 as a drug target as well as a tool for the diagnosis, treatment and development of p11/5-HT receptor related disorders. The invention further relates to p11 knock-out animals as well as p11 transgenic animals and their use as models for the development of novel psychotherapeutic agents, and to methods of diagnosis, prophylaxis and treatment of p11/5-HT receptor related disorders.

This application claims the benefit of U.S. Provisional Applications60/813,170 filed Jun. 13, 2006 and 60/878,730 filed Jan. 5, 2007, thecontents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention introduces a biological association between p11and 5-HT_(1B) and 5-HT₄ receptors, providing the use of p11 as a drugtarget for diseases involving these receptors, as well as a diagnostictool for the identification of patients suffering from p11/5-HT receptorrelated disorders.

BACKGROUND OF THE INVENTION

Currently available anti-depressant and anti-anxiety drugs target thebio-synthetic, degradative and operative pathways of monoamineneurotransmitters such as norepinephrine, dopamine and, in particular,serotonin (5-hydroxy-tryptamine or 5-HT). Serotonin, first discovered inthe late 1940's, plays a crucial role in modulating numerous functionsin the body including mood, sleep, appetite and sexual activities. Itfunctions both as a neurotransmitter within the central nervous systemand also as a peripheral signal modulator. Consequently, alterations inserotonin availability and activity have been linked to depression,eating disorder (e.g. bulimia), obsessive compulsive disorders (OCD),drug addiction, attention deficit disorder (ADD), attention deficithyperactive disorder (ADHD), premenstrual syndrome, anxiety disorders,aggression, sleep disorders, sexual dysfunction, gastrointestinaldisorders (e.g. irritable bowel syndrome), mania, migraine, and bipolardisorder. Conventional anti-depressants typically regulate the signaltransmission by either (1) preventing the degradation of serotonin byinhibiting monoamine oxygenase or (2) increasing neuronal transport ofserotonin by inhibiting serotonin re-uptake by the presynaptic neurons.Despite over half a century of intensive study of serotonin pathways,however, the understanding of these pathways is incomplete, and thereare no established biochemically-based diagnostics or biomarkers forserotonin pathway dysfunction.

5-HT (serotonin) receptors are heterogeneous and are found on thesurface of a variety of cells. The 5-HT_(D3) receptor is one of 14serotonin receptor subtypes and is found abundantly throughout thecentral nervous system. The structure, distribution and apparentfunction of 5-HT_(1B) receptors are very similar in rodents and humans.This receptor has been linked to a diverse range of physiologicfunctions and behaviors including mood, cognition, aggression,addiction, sleep and feeding. 5-HT_(1B) receptors are found both onserotonin- and nonserotonin-containing neurons. 5-HT_(1B) receptors arefound predominantly on the pre-synaptic portion of the neuron, wherethey function as terminal autoreceptors involved in the regulation ofserotonin release by neurons. When stimulated by binding to serotonin,they inhibit the release of additional serotonin by the neuron; when notstimulated, serotonin release is enhanced. Blocking of these 5-HT_(1B)receptors thus tends to enhance serotonin levels. There is some evidencethat 5-HT_(1B) receptors are heteroreceptors, involved in controllingthe release of other neurotransmitters, such as acetylcholine,glutamate, dopamine, norepinepherine and gamma-aminobutyric acid, aswell as serotonin. Some 5-HT_(1B) receptors are also foundpost-synaptically. The 5-HT₄ receptor is found in the gastrointestinalsystem, where it is involved in gastrointestinal motility, as well asthe central nervous system. Whereas 5-HT_(1B) is generally associatedwith a decrease in cAMP, the 5-HT₄ receptor is associated with increasedcAMP activity. 5-HT₄ receptors in the brain modulate neurotransmitter(acetylcholine, dopamine, serotonin and GABA) release and enhancesynaptic transmission. They may also play a role in memory enhancement,by promoting release of non-amyloidogenic soluble amyloid precursorprotein (sAPPalpha). As Alzheimer's disease is widely thought to bemediated by deposition of beta-amyloid plaque formation, enhancing 5-HT₄receptor function, thereby enhancing release of sAPPalpha, represents apotential approach to treatment or prophylaxis of Alzheimer's disease.

The protein p11 is a member of the S100 EF-hand protein family. p11 isalso known as annexin-II light chain, lipocortin-II light chain,calpactin I light chain, 42C, or 5-100 related protein, and these termsmay be used interchangeably herein. R. Donato, Biochim. Biophys. Acta,1450, 191 (1999). It is present in a variety of cells separately or as aheterotetramer. The heterotetramer is composed of two copies of p36,also known as annexin-II or calpactin-I heavy chain, and two copies ofp11. Within the cell, the heterotetramer is localized at the cytoplasmicsurface of the plasma membrane in the submembranous cytoskeleton, and itis suggested that the complex may play a role in membrane traffickingevents such as exocytosis, endocytosis and cell-cell adhesion. p11 hasbeen claimed to have a role in tumor cell invasion, tumor growth, andmetastasis. US 2004/0142897A1. p11 has not previously been identified asbeing involved with 5-HT receptors or psychiatric disorders.

SUMMARY OF THE INVENTION

Applicants have now surprisingly discovered that p11 protein interactsspecifically with 5-HT_(1B) receptors and appears to help regulatesignaling of the brain messenger chemical serotonin, a key target ofmany psychotherapeutic agents. Svenningsson et al., Science (2006)331:77-80; Svenningsson et al., Current Opinion in Pharmacology (2006),6:1-6 (both incorporated herein by reference). p11 appears to play acrucial role in the recruitment of 5-HT_(1B) receptors to the neuronalplasma membrane where they are more functional. Applicants have furtherdiscovered that p11 also interacts with 5-HT₄ receptors. Applicants haveshown that p11 levels may be directly involved in the development ofdepression, anxiety disorders and similar psychiatric illnesses that arethought to involve faulty serotonin receptors. Comparison of p11 levelsin the brains of depressed subjects (depressed humans and mice models)to those of non-depressed subjects (non-depressed humans and controlmice) shows a substantially lower level of p11 in depressed subjectscompared to non-depressed subjects. Moreover, p11 levels tend to behigher in subjects treated with various types of antidepressants,including tricyclic antidepressants, monoamine oxidase inhibitors(MAOIs) and electroconvulsive therapy. There is an over-expression ofp11 in animals that are treated with anti-depressants. For example, wehave observed that monkeys receiving the selective serotonin reuptakeinhibitor fluoxetine display a significant (more than twofold) increasein p11 expression in peripheral blood mononuclear cells (PBMC), andsimilar effects are demonstrated in the brains of mice receivingfluoxetine. Similarly, animal models with a p11 knock-out gene exhibitfewer 5-HT_(1B) receptors at the neuronal plasma membrane, have reducedserotonin signaling, and exhibit a depression-like phenotype.Interestingly, pill expression decreases in response to excess levels ofglucocorticoid hormones, which are often released in response to stress,which in light of Applicants' work, provides a possible biochemicalexplanation for the observed link between depression and highlystressful events. Based on these surprising discoveries by Applicants,p11 is shown to be a suitable diagnostic target as well as a drug targetand screening tool for the development of treatments for disorderspreviously associated with 5-HT_(1B) or 5-HT₄ receptors, or withserotonin function (or lack thereof).

The term “p11/5-HT receptor related disorders” as used herein includeany disorders mediated by, associated with, caused by, affected by,triggered by or involving mobilization (or lack of mobilization) of 5-HTreceptors, e.g., 5-HT_(1B) or 5-HT₄ receptors, by the p11 protein.p11/5-HT receptor related disorders may include, but are not limited to,psychiatric disorders (e.g. depression, anxiety disorders, aggression,mania, bipolar disorder, attention deficit disorder, attention deficithyperactive disorder, drug addiction and obsessive compulsive disorder,and Alzheimer's disease) sleep disorders (e.g. insomnia), eatingdisorders (e.g. bulimia), sexual dysfunction, and gastrointestinaldisorders (e.g. irritable bowel syndrome); especially depression.

The invention thus provides, inter alia:

-   -   1. Methods of diagnosing p11/5-HT receptor related disorders;    -   2. Methods of identifying compounds useful in treating p11/5-HT        receptor related disorders;    -   3. Transgenic animals which over- or under-express p11; and    -   4. Methods of treating p11/5-HT receptor related disorders.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the invention relates to a method of diagnosingp11/5-HT receptor related disorders in a subject comprising (1) assayingthe level of p11 protein in a biological sample of said subject, e.g., ablood or tissue sample, for example monocytes and/or leukocytes, e.g.,peripheral blood mononuclear cells (PBMC), and (2) comparing said p11level to a reference standard, e.g., the p11 level in a controlpopulation that does not have or is not suspected of having any p11/5-HTreceptor related disorders, wherein an abnormal level of p11 in asubject compared to the reference standard constitutes a positivediagnosis of p11/5-HT receptor related disorders (Method 1).

Thus the invention includes the following embodiments of Method 1

-   1.1. The method of Method 1 wherein the p11/5-HT receptor related    disorder is a disorder associated with an abnormally low level of    p11, wherein a reduced level of p11 in a subject compared to the    reference standard constitutes a positive diagnosis of such    disorders.-   1.2. A method according to method 1 or 1.1, wherein the disorder is    associated with an abnormally low level of p11 and is selected from    a group consisting of depression, obsessive compulsive disorder,    drug addiction, eating disorders, attention deficit disorder and    attention deficit hyperactive disorder.-   1.3. A method according to any of the previous methods wherein the    disorder is associated with an abnormally low level of p11 and is    depression.-   1.4. A method according to method 1 wherein said p11/5-HT receptor    related disorders are disorders associated with an abnormally high    level of p11, wherein an elevated level of p11 in a subject compared    to that of the reference standard constitutes a positive diagnosis    of such disorders.-   1.5. A method according to any of method 1 or 1.4 wherein the    disorder is associated with an abnormally high level of p11 and is    selected from a group consisting of mania, bipolar disorder, anxiety    disorders, aggression, sleep disorders, sexual dysfunction and    gastrointestinal disorders.-   1.6. The method according to any of the foregoing methods wherein    said level of pH is determined by assaying p11 protein level in    peripheral blood mononuclear cells (PBMC) from said subject.-   1.7. The method according to 1.6 wherein the PBMCs are selected from    monocytes, NK cells, and CD-8+ T-cells.-   1.8. The method according to any of the foregoing methods, wherein    said level of p11 is determined by assaying p11 mRNA level in a    biological sample from said subject.-   1.9. The method according to any of the foregoing methods wherein    the level of p11 is determined using a monoclonal antibody specific    for p11.-   1.10. The method according to any of the foregoing methods wherein    the p11/5-HT receptor related disorder is a p11/5-HT_(1B) receptor    related disorder.-   1.11. The method according to any of the foregoing methods wherein    the p11/5-HT receptor related disorder is a p11/5-HT₄ receptor    related disorder.-   1.12. A kit useful in measuring p11 levels, e.g. in accordance with    any of the foregoing methods 1-1.11, comprising an oligonucleotide    probe specific for p11 mRNA or a monoclonal antibody specific for    p11, and instructions for use.-   1.13. Use of an oligonucleotide probe specific for p11 mRNA or a    monoclonal antibody specific for p11 in any of methods 1-1.11.-   1.14. Use of an oligonucleotide probe specific for p11 mRNA or a    monoclonal antibody specific for p11 in the manufacture of a reagent    for use in a method according to any of claims 1-1.11 or in a kit    according to 1.12.

In another embodiment, the invention relates to a method to identify p11modulators useful to treat or ameliorate p11/5-HT receptor relateddisorders comprising assaying for the ability of a candidate modulatorto regulate (either up or down) p11 expression or p11 activitiesassociated with 5-HT receptors (method 2).

Therefore, method 2 includes

-   2.1. A method to identify p11 modulators useful to treat or    ameliorate p11/5-HT receptor related disorders comprises the steps    of providing a first sample and a second sample, e.g., a cell    culture or cell or tissue sample, containing equivalent amounts of    p11 gene product (e.g., protein or mRNA); contacting the first    sample with the candidate p11 modulator; and determining whether the    amounts of p11 gene product in the first sample has changed, wherein    an increased amount of gene product indicate that the candidate    modulators can be useful to treat or ameliorate disorders associated    with abnormally low level of p11 while a decreased amount indicates    that the candidate modulators can be useful to treat or ameliorate    disorders associated with abnormally low level of p11.-   2.2. A method to identify p11 modulators useful to treat or    ameliorate p11/5-HT receptor related disorders comprises the steps    of providing a first sample and a second sample containing    equivalent number of 5-HT_(1B) and/or 5-HT₄ receptors at cell    surface; contacting the first sample with the candidate p11    modulator; and determining whether the number of 5-HT_(1B) and/or    5-HT₄ receptors at cell surface of the first sample has changed    relative to the second sample, wherein an increased number of    5-HT_(1B) and/or 5-HT₄ receptors at cell surface indicate that the    candidate modulators can be useful to treat or ameliorate disorders    associated with abnormally low level of p11 while a decreased number    of 5-HT₁₁₃ and/or 5-HT₄ receptors at cell surface indicates that the    candidate modulators can be useful to treat or ameliorate disorders    associated with abnormally low level of p11.-   2.3. A method to identify p11 modulators comprising contacting a    candidate p11 modulator with a cell comprising a reporter gene    operably linked to a p11 promoter, and using the reporter gene    expression level as a proxy for p11 expression.-   2.4. A method to identify p11 modulators useful to treat or    ameliorate disorders associated with low levels of p11, comprising    assaying for the ability of a candidate modulator to up-regulate p11    expression or increase p11 activities associated with 5-HT_(1B)    and/or 5-HT₄ so as to recruit 5-HT_(1B) and/or 5-HT₄ receptors at    the neuronal plasma membrane.-   2.5. A method according to any of methods 2-2.4 wherein the    disorders associated with abnormally low levels of p11 are selected    from depression, obsessive compulsive disorder, drug addiction,    eating disorders, attention deficit disorder or attention deficit    hyperactive disorder, and Alzheimer's disease.-   2.6. A method according to any of the preceding methods 2-2.5    wherein the disorder is depression.-   2.7. A method according to any of the preceding methods 2-2.6    wherein a modulator of p11 is used as a positive control.-   2.8. A method according to method 2.6 wherein the modulator of p11    is selected from tricyclic antidepressants, selective serotonin    reuptake inhibitors, triptans, and monoamine oxidase inhibitors.-   2.9. A method according to method 2.8 wherein the modulator of p11    is a tricyclic antidepressant selected from amitriptyline (brand    name: Elavil), desipramine (brand name: Norpramin), imipramine    (brand name: Tofranil), and nortriptyline (brand name: Aventyl,    Pamelor)-   2.10. A method according to method 2.8 wherein the modulator of p11    is imipramine.-   2.11. A method according to method 2.8 wherein the modulator of p11    is a Monoamine Oxidase Inhibitors (MAOI), e.g., selected from    isocarboxazid (brand name: Marplan); phenelzine (brand name: Nardil)    and tranlcypromine (brand name: Parnate)-   2.12. A method according to 2.11 wherein the MAOI is tranicypromine.-   2.13. A method according to method 2.8 wherein the modulator of p11    is a selective serotonin reuptake inhibitor, e.g., selected from    citalopram (brand name: Celexa); escitalopram (brand name: Lexapro);    fluoxetine (brand name: Prozac); paroxetine (brand names: Paxil,    Pexeva); sertraline (brand name: Zoloft).-   2.14. A method to identify p11 modulators useful to treat or    ameliorate disorders associated with high levels of p11, comprising    assaying for the ability of a candidate modulator to down-regulate    p11 expression or inhibit or reduce p11 activities associated with    5-HT_(1B) and/or 5-HT₄ so as to reduce or inhibit p11's ability to    recruit 5-HT_(1B) and/or 5-HT₄ receptors to neuronal plasma    membrane.-   2.15. A method according to 2.14 wherein the disorders associated    with high levels of p11 include, but are not limited to mania,    dipolar disorder, anxiety disorders, aggression, sleep disorders,    sexual dysfunction and gastrointestinal disorders.-   2.16. A method according to 2.15 to identify p11 modulators useful    to treat or ameliorate disorders associated with high levels of p11,    wherein said modulators are selected from siRNA, antisense    oligonucleotides, and monoclonal antibodies to p11.-   2.17. A method according to any of methods 2-2.16 wherein p11    suppressor compounds selected from siRNA, antisense    oligonucleotides, and monoclonal antibodies to p11 are used as    controls or comparators.-   2.18. A method e.g according to any of method 2-2.17 to identify p11    mimetics useful to treat or ameliorate disorders associated with    abnormally low level of p11, comprising assaying for the ability of    a candidate p11 mimetic to associate or interact with 5-HT_(1B)    and/or 5-HT₄ receptors so as to recruit 5-HT_(1B) and/or 5-HT₄    receptors to the neuronal plasma membrane.-   2.19. A method according to method 2.18 to identify p11 mimetics    useful to treat or ameliorate depression comprising assaying for the    ability of a candidate p11 mimetic to recruit 5-HT_(1B) and/or 5-HT₄    receptors to the neuronal plasma membrane.-   2.20. The method according to any of the foregoing methods wherein    the p11/5-HT receptor related disorder is a p11/5-HT_(1B) receptor    related disorder.-   2.21. The method according to any of the foregoing methods wherein    the p11/5-HT receptor related disorder is a p11/5-HT₄ receptor    related disorder.-   2.22. A cell comprising a reporter gene operably linked to a p11    promoter.-   2.23. Use of a cell according to 2.22 in a method according to any    of methods 2-2.21.

In yet another embodiment, the invention is directed to a transgenicnon-human mammal or progeny thereof, which over- or underexpresses p11,and their use in methods to discover new pharmaceuticals (method 3). Theinvention thus includes

-   3.1. A p11 knock-out non-human mammal, wherein said non-human mammal    possesses a DNA sequence that has a defect, mutation or deficiency    in the p11 gene and therefore under-expresses p11 proteins and/or    possesses fewer 5-HT_(1B) and/or 5-HT₄ receptors at the neuronal    plasma membrane and therefore exhibits a depression-like phenotype    compared to a wild-type nonhuman mammal of the same species.-   3.2. A p11 transgenic non-human mammal, wherein said non-human    mammal over-expresses p11 proteins and/or exhibits an elevated level    of 5-HT_(1B) and/or 5-HT₄ receptors at the neuronal plasma membrane    and therefore exhibits a hyperactive phenotype compared to a    wild-type non-human mammal of the same species.-   3.3. A non-human mammal according to 3.1 or 3.2 which is.a mouse.-   3.4. A non-human mammal, e.g. according to 3.2 or 3.3, having a    transgene comprising a coding region encoding p11, operably linked    to a regulatable promoter, e.g. the doxycycline-regulatable    calcium/calmodulin-dependent protein kinase II (CamKII) promoter.-   3.5. A method to study p11/5-HT receptor related disorders or to    develop novel psychotherapeutic agents useful to treat or ameliorate    p11/5-HT receptor related disorders comprising (1) administering    said agents to (a) p11 knock-out mammals according 3.1 or 3.3    and (b) control mammals of the same species that do not have or are    not suspected of having any p11/5-HT receptor related disorders;    and (2) assessing and comparing the behavior and/or levels of    5-HT_(1B) or 5-HT₄ receptor of said (a) knock-out mammals and (b)    control mammals.-   3.6. A method, e.g. according to 3.4, to study depression comprising    administering an antidepressant to (a) p11 knock-out mammals and (b)    control mammals that do not have or is not suspected of having    depression; and (2) assessing and comparing the behavior and/or    5-HT_(1B) or 5-HT₄ receptor levels of said (a) knock-out mammals    and (b) control mammals.-   3.7. A p11 mouse model useful for developing novel anti-psychotic    agents comprising (1) administering said agents to (a) p11    over-expressing mice and (b) control mice that do not have or is not    suspected of having any p11/5-HT receptor related disorders; and (2)    assessing and comparing the p11 levels of said (a) transgenic mice    and (b) control mice.-   3.8. A method to study p11/5-HT receptor related disorders or to    develop novel psychotherapeutic agents useful to treat or ameliorate    p11/5-HT receptor related disorders comprising (1) administering    said agents to (a) mammals according 3.2 or 3.3 and (b) control    mammals of the same species that do not have or are not suspected of    having any p11/5-HT receptor related disorders; and (2) assessing    and comparing the p11 levels of said (a) mammals according to 3.2 or    3.3 and (b) control mammals.-   3.9. A p11 mouse model useful for developing novel anti-psychotic    agents comprising (1) administering said agents to (a) p11    over-expressed mice, e.g. according to 3.2 and (b) control mice that    do not have or is not suspected of having any p11/5-receptor related    disorders; and (2) assessing and comparing the behavior and/or p11    or 5-HT receptor levels of said (a) transgenic mice and (b) control    mice.-   3.10. The method or model according to any of the foregoing methods    or models wherein the 5-HT receptor being measured or being over- or    under-expressed is the 5-HT_(1B) receptor.-   3.11. The method or model according to any of the foregoing methods    or models wherein the 5-HT receptor being measured or being over- or    under-expressed is the 5-HT₄ receptor.

The invention further relates to a method to treat a patient sufferingfrom a p11/5-HT receptor related disorder, comprising administration ofa therapeutically effective amount of a p11 modulator (method 4).

-   4.1. Method 4 when the patient is first identified according to any    of methods 1-1.11.-   4.2. Method according to 4 or 4.1 wherein the p11/5-HT receptor    related disorder is a disorder associated with abnormally low levels    of p11, e.g., selected from depression, obsessive compulsive    disorder, drug addiction, eating disorders, attention deficit    disorder or attention deficit hyperactive disorder, and Alzheimer's    disease.-   4.3. Method according to 4.2 wherein the disorder is depression.-   4.4. Method according to any of the previous methods 4-4.3    comprising administration of a p11 modulator identified in    accordance with method 3, e.g. using any of embodiments 3.1-3.11.-   4.5. A method according to any of the previous methods wherein the    modulator of p11 is selected from tricyclic antidepressants,    selective serotonin reuptake inhibitors, and monoamine oxidase    inhibitors.-   4.6. A method according to method 4.4 wherein the modulator of p11    is a tricyclic antidepressant selected from amitriptyline (brand    name: Elavil), desipramine (brand name: Norpramin), imipramine    (brand name: Tofranil), and nortriptyline (brand name: Aventyl,    Pamelor)-   4.7. A method according to method 4.5 wherein the modulator of p11    is imipramine.-   4.8. A method according to method 4.4 wherein the modulator of p11    is a Monoamine Oxidase Inhibitors (MAOI) selected from isocarboxazid    (brand name: Marplan); phenelzine (brand name: Nardil) and    tranlcypromine (brand name: Parnate)-   4.9. A method according to 4.8 wherein the MAOI is tranlcypromine-   4.10. A method according to method 4.4 wherein the modulator of p11    is a selective serotonin reuptake inhibitor selected from citalopram    (brand name: Celexa); escitalopram (brand name: Lexapro); fluoxetine    (brand name: Prozac); paroxetine (brand names: Paxil, Pexeva);    sertraline (brand name: Zoloft).-   4.11. A method according to any of the previous methods 4-4.10 to    treat or ameliorate in a subject suffering from p11/5-HT receptor    related disorders comprising administering to said subject an    effective amount of p11 modulator or mimetic so as to regulate (up    or down) p11 expression and/or 5-HT_(1B) or 5-HT₄ receptors at the    neuronal plasma membrane.-   4.12. A method according to 4.11 treat or ameliorate in a subject    suffering from disorders associated with abnormally low level of p11    comprising administering to said subject an effective amount of p11    modulator or mimetic so as to up-regulate p11 expression or recruit    5-HT_(1B) or 5-HT₄ receptors to the neuronal plasma membrane.-   4.13. A method according to 4.12 wherein the disorder is selected    from depression, obsessive compulsive disorder, drug addiction,    eating disorders, attention deficit disorder or attention deficit    hyperactive disorder, and Alzheimer's disease.-   4.14. The method according to any of the foregoing methods wherein    the p11/5-HT receptor related disorder is a p11/5-HT_(1B) receptor    related disorder.-   4.15. The method according to any of the foregoing methods wherein    the p11/5-HT receptor related disorder is a p11/5-HT₄ receptor    related disorder.-   4.16. The use of a p11 modulator, e.g., as herein described, in the    manufacture of a medicament to treat a p11/5-HT receptor mediated    disorder, e.g., in any of methods 4-4.15.-   4.17. A pharmaceutical composition comprising a p11 modulator for    use in treating a p11/5-HT receptor mediated disorder, e.g., in any    of methods 4-4.15.

In another embodiment, the invention relates to a method to enhance p11expression or upregulate p11 in a patient suffering from a p11/5-HTreceptor related disorder, comprising the administration of a nucleicacid expressing p11 or inducing the expression of p11 in the brain ofsaid patient, wherein said nucleic acid up-regulates p11 or increasesp11 expression in the brain of said patient (Method 5). The nucleic acidencoding or inducing the expression of p11 is preferably delivered tothe brain of the patient by a vector or by cells comprising the nucleicacid. Such vectors may be in the form of DNA or RNA in a suitabledelivery system, for example liposome-encapsulated DNA constructs or inthe form of a viral vector, for example a replication deficientadenoviral vector or adeno-associated viral vector. In a preferredembodiment, the vector is a viral vector which provides transientexpression of the desired transgene rather than integration of thetransgene into the infected cell, for example a vector derived fromadenovirus. The vector may for example comprise DNA encoding p11operably linked to a promoter, e.g., a constitutive promoter (e.g., suchas the CMV promoter), a tissue-specific promoter (e.g., such as theneuron-specific enolase (NSE) pronloter) or an inducible promoter (e.g.,a tetracycline or doxycycline inducible promoter), that controls theexpression of the DNA encoding p11 so as to enable the expression of p11in. the target cell. For example, a viral vector may be a recombinantlymodified adenovirus comprising a p11 DNA construct under control of atissue-specific promoter and lacking a functional copy of one or moregenes essential for replication (for example, the E1 and/or E3 gene),such that the virus can replicate only in a helper cell line or otherenvironment where the product of the gene or genes essential forreplication is supplied. Viral vectors may also comprise surfacemodifications to reduce immunogenicity and/or to target the vectors tothe desired cells. Viral vectors suitable for gene therapy in the CNSare known in the art, e.g., including vectors targeted to CNS cells andvectors utilizing tissue specific or inducible promoters for expressionof the transgene, for example as described in Benitez, et al., “GeneTherapy Targeting in Central Nervous System”, Current Gene Therapy(2003) 3: 127-145 (incorporated herein by reference). Non-viral nucleicacid delivery systems may comprise nucleic acid associated or complexedwith agents to facilitate the entry of the nucleic acid across thecellular membrane. Examples of such non-viral vector complexes includethe formulation with polycationic agents which facilitate thecondensation of the DNA and lipid-based delivery systems, for example aliposome based delivery system. In another embodiment, said nucleic acidexpressing or inducing the expression of p11 is delivered by a cell, forexample a neuronal stem cell which expresses p11 at levels higher thanthe levels expressed by the patient's cells, which is transplanted intothe brain of the patient. The source of the cell may be the patient or ahuman donor. The p11 expression in the transplanted cell may be achievedby selection for p11 expression and/or by transformation, e.g., ex vivotransformation, with (i) a p11 construct, (ii) a promoter upstream ofthe native p11 sequence which enhances p11 expression, or (iii) aconstruct encoding a protein which enhances p11 expression. The cell ispreferably stably transformed, e.g., using a retroviral or lentiviralvector, so that the construct is integrated into the genome of the cell.The cellular or viral vectors may further comprise a “safety gene” forexample the herpes simplex virus thymidine kinase gene (HSV-tk) whichmakes a host cell susceptible to gancyclovir, so that the cells orviruses can be easily destroyed if the cells become cancerous or thevirus infects the patient or there is otherwise a risk of harm to thepatient. In a preferred embodiment, the nucleic acid enhancing p11expression (for example a cell or viral vector comprising a constructexpressing p11) is introduced into the targeted cell via intracerebraladministration, e.g., into the hippocampus region. The nucleic acid maybe administered in a single dose or a multiplicity of treatments.

Therefore, the invention further provides methods as follows:

-   5.1 Method 5, wherein said method increases p11 expression.-   5.2 Method 5 or 5.1, wherein the nucleic acid construct is capable    of expressing p11 in a brain cell.-   5.3 Method 5, 5.1-5.2, wherein the p11/5-HT receptor related    disorder is a disorder associated with abnormally low levels of p11,    e.g., selected from depression, obsessive compulsive disorder, drug    addiction, eating disorders, attention deficit disorder or attention    deficit hyperactive disorder, and Alzheimer's disease.-   5.4 Method 5, 5.1-5.2, wherein the p11/5-HT receptor related    disorder is anxiety disorder or depression.-   5.5 Method 5, 5.1-5.2 wherein the p11/5-HT receptor related disorder    is depression.-   5.6 Method 5, 5.1 or 5.5, wherein said nucleic acid encoding p11 is    delivered by a replication-deficient adenoviral vector comprising    DNA encoding p11.-   5.7 Method 5.6, wherein the DNA encoding p11 is under control of a    tissue specific promoter, for example the NSE promoter.-   5.8 Method 5, 5.1 or 5.5, wherein said nucleic acid encoding p11 is    delivered by a stem cell transformed with a p11 construct.-   5.9 Any of Methods 5 or 5.1-5.8, wherein said nucleic acid of pit is    introduced via intracerebral administration.-   5.10 Any of methods 5 or 5.1-5.8, wherein said nucleic acid of p11    is introduced to the hippocampus.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. Thus, for example, reference to the “antibody” is a referenceto one or more antibodies and equivalents thereof known to those skilledin the art, and so forth. Treatment of “a” p11/5-HT_(1B) receptormediated disorder may include treatment of multiple such disorders.

The term “p11” herein refers to any and all forms of p11 polypeptide,including but not limited to, partial forms, isoforms, precursor forms,full length polypeptide, fusion proteins containing the p11 sequence orfragments of any of the above, from human or any other species.

The phrase “p11/5-HT receptor related disorders” herein refers to anydisorders mediated by, associated with, caused by, affected by,triggered by or involving p11 protein and its mobilization of 5-HTreceptors, especially the 5-HT_(1B) or 5-HT₄ receptors. p11/5-HTreceptor related disorders may include, but are not limited to,psychiatric disorders (e.g. depression, anxiety disorders, aggression,mania, bipolar disorder, attention deficit disorder, attention deficithyperactive disorder, Alzheimer's disease, drug addiction and obsessivecompulsive disorder) sleep disorders (e.g. insomnia), eating disorders(e.g. bulimia), sexual dysfunction, and gastrointestinal disorders (e.g.irritable bowel syndrome). “Disorders associated with abnormally lowlevel of p11” herein refers to disorders such as depression, obsessivecompulsive disorders, drug addiction, eating disorders, attentiondeficit disorder or attention deficit hyperactive disorder, orAlzheimer's disease, especially depression. Likewise, “disordersassociated with abnormally high level of p11” refers to disorders suchas mania, dipolar disorder, anxiety disorders, aggression, sleepdisorders, sexual dysfunction and gastrointestinal disorders.

The phrase “p11 modulator” refers to any substance or compound (e.g.small molecules or polypeptides as described herein) or methods oftreatment (e.g., electroconvulsive therapy) capable of changing (eitherincreasing or decreasing) expression of a gene encoding p11 protein,transcription of a p11 gene or cDNA into an mRNA, the translation of ap11 mRNA into protein, post-translational modification of a p11 protein,cellular or extracellular localization of a p11 protein, or amount ofp11 localized in or on the cell membrane or inside the cell, relative tothe p11 activity in similar cells. The term “p11 modulator” also refersto any substance capable of affecting (either positively or negatively)the ability of p11 proteins to recruit 5-HT_(1B) receptors to theneuronal plasma membrane. Examples of p11 modulators useful to treatdisorders associated abnormally low level of p11 such as depressioninclude tricyclic antidepressants (e.g. imipramine (Tofranil®),amitriptyline (ELAVIL®, ENDEP® TRYPTANOL®), clomipramine (ANAFRANIL®),desipramine (NORPRAMIN®, PERTOFRANE®), lofepramine (GAMANIL®, LOMONT®),nortriptyline (PAMELOR®), trimipramine (SURMONTIL®)). Other modulatorsuseful to treat or ameliorate disorders associated with low level of p11(e.g. depression) include Monoamine Oxidase Inhibitors (MAOI) (e.g.Tranylcypromine (Parnate), Isocarboxazid (Marplan), Moclobemide(Aurorix, Manerix, MOCLODURA®) or Phenelzine (Nardil)), and selectiveserotonin reuptake inhibitors (e.g., citalopram (brand name: Celexa);escitalopram (brand name: Lexapro); fluoxetine (brand name: Prozac);paroxetine (brand names: Paxil, Pexeva); sertraline (brand name:Zoloft)).

Conventional screening assays (both in vitro and in vivo) may be used toidentify modulators that inhibit or induce p11 activity and/or p11 geneexpression. One such assay is a gene reporter assay, wherein cellstransfected with a reporter construct comprising a marker gene (e.g.,luciferase or green fluorescent protein (GFP)) downstream of a p11binding site are contacted with a candidate modulator compound and thechanges in the expression of the marker protein is measured and comparedto a transfected cell sample that is not contacted with any modulator.Candidate modulators that either inhibit or induce marker proteinexpression are identified as drugs useful for the treatment of p11/5-HTreceptor related disorder. Candidate modulators that inhibit markerprotein expression would be useful drug candidates for the treatment ofdisorders associated with abnormally high level of p11 while a candidatemodulators that induce marker protein expression would be useful drugcandidates for the treatment of disorders associated with abnormally lowlevel of p11.

p11 modulators may include, e.g., natural or unnatural chemicalcompounds, in free or pharmaceutically acceptable salt form, sense orantisense p11 oligonucleotides, inhibitory antibodies to p11,p11-receptor blocking peptides, p11 antagonists, si RNA, triple helixDNA, ribozymes, RNA aptamers and/or double stranded RNA. The term“antisense” as used herein, refers to nucleotide sequences which arecomplementary to a specific DNA or RNA sequence. Therefore, “p11antisense polynucleotide” refers to any nucleotide sequence that iscomplementary to p11 DNA or RNA sequence. Functionally, p11 antisensepolynucleotide is capable of decreasing the expression of p11 protein ina cell. The term “antisense strand” is used in reference to a nucleicacid strand that is complementary to the “sense’ strand. Antisensemolecules may be produced by any method, including synthesis by ligatingthe gene(s) of interest in a reverse orientation to a viral promoterwhich permits the synthesis of a complementary strand. Once introducedinto a cell, this transcribed strand combines natural sequences producedby the cell to form duplexes. These duplexes then block either thefurther transcription or translation. The designation “negative” issometimes used in reference to the antisense strand, and “positive” issometimes used in reference to the sense strand. Similarly, the term“sense” as used herein, refers to nucleotide sequences which can betranslated to produce a specific polypeptide or fragment thereof.Therefore, “p11 sense polynucleotide” refers to any nucleotide sequencethat can be translated to produce p11 polypeptide or fragment thereof.Functionally, p11 sense polynucleotide is capable of increasing theexpression of p11 proteins in a cell.

Specifically, substances that inhibit the expression of p11 at thenucleic acid level may include ribozymes, antisense oligonucleotides,triple helix DNA, RNA aptamers and/or double stranded RNA directed to anappropriate nucleotide sequence of the p11 nucleic acid. Theseinhibitory molecules may be created using conventional techniques by oneof skill in the art without undue burden or experimentation. Forexample, modifications (e.g. inhibition) of gene expression can beobtained by designing antisense molecules, DNA or RNA, to the controlregions of the genes encoding the polypeptides discussed herein, i.e. topromoters, enhancers, and introns. For example, oligonucleotides derivedfrom the transcription initiation site, e.g., between positions −10 and+10 from the start site may be used. Notwithstanding, all regions of thegene may be used to design an antisense molecule in order to createthose which gives strongest hybridization to the mRNA and such suitableantisense oligonucleotides may be produced and identified by standardassay procedures familiar to one of skill in the art.

Similarly, inhibition of the expression of gene expression may beachieved using “triple helix” base-pairing methodology. Gee, J. E. etal. (1994) In: Huber, B. E. and B. I. Can, Molecular and ImmunologicApproaches, Futura Publishing Co., Mt. Kisco, N.Y.). These molecules mayalso be designed to block translation of mRNA by preventing thetranscript from binding to ribosomes. Ribozymes, enzymatic RNAmolecules, may also be used to inhibit gene expression by catalyzing thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered “hammerhead” or “hairpin” motifribozyme molecules that can be designed to specifically and efficientlycatalyze endonucleolytic cleavage of gene sequences, for example, thegene for p11. Specific ribozyme cleavage sites within any potential RNAtarget are initially identified by scanning the target molecule forribozyme cleavage sites which include the following sequences: GUA, GUUand GUC. Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Grassi andMarini, 1996, Annals of Medicine 28: 499-510; Gibson, 1996, Cancer andMetastasis Reviews 15: 287-299; Cotten et al, 1989 EMBO J. 8:3861-3866.RNA aptamers can also be introduced into or expressed in a cell tomodify RNA abundance or activity. RNA aptamers are specific RNA ligandsfor proteins, such as for Tat and Rev RNA (Good et al., 1997, GeneTherapy 4: 45-54) that can specifically inhibit their translation. Genespecific inhibition of gene expression may also be achieved usingconventional double stranded RNA technologies. A description of suchtechnology may be found in WO 99/32619 which is hereby incorporated byreference in its entirety.

Antisense molecules, triple helix DNA, RNA aptamers and ribozymes of thepresent invention may be prepared by any method known in the art for thesynthesis of nucleic acid molecules. These include techniques forchemically synthesizing oligonucleotides such as solid phasephosphoramidite chemical synthesis. Alternatively, RNA molecules may begenerated by in vitro and in vivo transcription of DNA sequencesencoding the genes of the polypeptides discussed herein. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively, cDNAconstructs that synthesize antisense RNA constitutively or inducibly canbe introduced into cell lines, cells, or tissues.

siRNA molecules of the present invention can be generated by annealingtwo complementary single-stranded RNA molecules together (one of whichmatches a portion of the target mRNA) (Fire et al., U.S. Pat. No.6,506,559) or through the use of a single hairpin RNA molecule thatfolds back on itself to produce the requisite double-stranded portion(Yu et al. (2002) Proc. Natl. Acad. Sci. USA 99:6047-52). The siRNAmolecules can be chemically synthesized (Elbashir et al. (2001) Nature411:494-98) or produced by in vitro transcription using single-strandedDNA templates (Yu et al., supra). Alternatively, the siRNA molecules canbe produced biologically, either transiently (Yu et al., supra; Sui etal. (2002) Proc. Natl. Acad. Sci. USA 99:5515-20) or stably (Paddison etal. (2002) Proc. Natl. Acad. Sci. USA 99:1443-48), using an expressionvector(s) containing the sense and antisense siRNA sequences. Recently,reduction of levels of target mRNA in primary human cells, in anefficient and sequence-specific manner, is demonstrated using adenoviralvectors that express hairpin RNAs, which are further processed intosiRNAs (Arts et al. (2003) Genome Res. 13:2325-32).

As used herein, the phrase “p11 activities” refers to any directbiochemical activity of p11 or indirect activity associated with p11 soas to affect (positively or negatively) p11's interaction with 5-HT_(1B)receptors. Modulators that increase p11 activities with 5-HT_(1B)receptors may be any substance that increases the association of p11 to5-HT_(1B) receptors so as to increase the ability of p11 proteins torecruit 5-HT_(1B) receptors to the neuronal plasma membrane. Conversely,modulators that inhibit or reduce p11 activities with 5-HT_(1B)receptors may be any substance that blocks or reduces the interactionbetween p11 and 5-HT_(1B) receptors so as to reduce the ability of p11proteins to recruit 5-HT_(1B) receptors to the neuronal plasma membrane.

The term “p11 mimetic” refers to a natural or unnatural substance orpolypeptide or any fragment thereof that mimics p11 protein instructure, function, property and/or activity, thereby modulating,regulating or increasing 5-HT_(1B) receptor availability at the neuronalplasma membrane. p11 mimetic may mimic p11 in whole or in part.

The term “subject” refers to any human or nonhuman organism.

A “control subject” refers to any human or nonhuman organism that doesnot have and/or is not suspected of having a disorder, syndrome,disease, condition and/or symptom of p11/5-HT receptor relateddisorders.

The term “biological sample” may include any sample comprisingbiological material obtained from e.g. an organism, body fluid, wasteproduct, cell or part of a cell thereof, cell line, biopsy, tissueculture or other source containing a p11 protein, polypeptide,oligonucleotide, mRNA or polynucleotide or any fragment of any of theabove.

A “positive diagnosis” of a p11/5-HT receptor related disorder refers toa condition where the subject being examined exhibits an abnormal levelof p11 compared to control subject who does not have and/or is notsuspected of having a p11/5-HT receptor related disorder. Abnormal levelrefers to a level that is higher or lower than that in a controlsubject. For instance, a subject with a positive diagnosis of depressionexhibits a depressed level of p11 compared to a control subject who doesnot have and/or is not suspected of having depression and/or symptomthereof. On the other hand, a subject with a positive diagnosis ofanxiety disorders state exhibits an elevated p11 expression compared toa control subject who does not have and/or is not suspected of havinganxiety disorders and/or symptom thereof.

The level of p11 may be determined by assaying p11 proteins in a sampleof tissue or cells obtained from a subject of a type which expressesp11. For example, monocytes and/or lymphocytes may be used. Similarly,p11 level may also be determined by assaying for p11 mRNA level in thesample. p11 gene expression (e.g. mRNA levels) may be determined usingmethods familiar to one of skill in the art, including, for example,conventional Northern analysis or commercially available micro-arrays.Additionally, the effect of test compounds' inhibition of p11 and/orrelated regulatory protein levels can be detected with an ELISAantibody-based assay or fluorescent labeling reaction assay. An abnormallevel of p11 protein or mRNA in a subject compared to a reference, e.g.,a control subject or control population (or a reference standard basedon prior measurements in a control population) constitutes a positivediagnosis of p11/5-HT receptor related disorders. Therefore, an elevatedlevel of p11 in a subject compared to the reference constitutes apositive diagnosis of disorders associated with high levels of p11, e.g.mania, dipolar disorder, anxiety disorders, aggressive disorder, sleepdisorders, sexual dysfunction and gastrointestinal disorders (e.g. IBD).On the other hand, a depressed or reduced level of p11 in a subjectcompared to that in a control subject constitutes a positive diagnosisof disorders associated with low levels of p11, e.g. depression,obsessive compulsive disorders, drug addiction, eating disorders,attention deficit disorder or attention deficit hyperactive disorder. Ina preferred embodiment, the invention encompasses a method of diagnosingin a subject suffering from depression, comprising assaying p11 level insaid subject and comparing such level to the p11 level in a controlsubject, wherein a depressed level of p11 in said subject compared tothat in a control subject constitutes a positive diagnosis ofdepression.

As used herein, the term “antibody” refers to intact molecules as wellas fragments thereof, such as Fa, F(ab′)₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind p11 polypeptidescan be prepared using intact polypeptides or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptides orpeptides used to immunize an animal can be derived from the translationof RNA or synthesized chemically, and can be conjugated to a carrierprotein, if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin and thyroglobulin. The coupledpeptide is then used to immunize an animal (e.g., a mouse, a rat or arabbit).

Factors for consideration for optimizing a therapy for a patient includethe particular condition being treated, the particular mammal beingtreated, the clinical condition of the individual patient, the site ofdelivery of the active compound, the particular type of the activecompound, the method of administration, the scheduling ofadministration, and other factors known to medical practitioners. Thetherapeutically effective amount of an active compound to beadministered will be governed by such considerations, and is the minimumamount necessary for the treatment of p11 mediated disorders,preferably, depression.

Suitable antibodies to p11 or related regulatory proteins can beobtained from a commercial source or produced according to conventionalmethods. For example, described herein are methods for the production ofantibodies capable of specifically recognizing one or moredifferentially expressed gene epitopes. Such antibodies may include, butare not limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

For the production of antibodies to p11 polypeptides discussed herein,various host animals may be immunized by injection with thepolypeptides, or a portion thereof. Such host animals may include, butare not limited to, rabbits, mice, and rats. Various adjuvants may beused to increase the immunological response, depending on the hostspecies, including, but not limited to, Freund's (complete andincomplete), mineral gels such as aluminum hydroxide, surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentiallyuseful human adjuvants such as BCG (bacille Calmette-Guerin) andCorynebacterium parvum.

Polyclonal antibodies are heterogeneous populations of antibodymolecules derived from the sera of animals immunized with an antigen,such as target gene product, or an antigenic functional derivativethereof. For the production of polyclonal antibodies, host animals suchas those described above, may be immunized by injection with thepolypeptides, or a portion thereof, supplemented with adjuvants as alsodescribed above.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture, such techniques being well known in the art. Such antibodiesmay be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD andany subclass thereof, preferably IgG. The hybridoma or transformed cellline producing the mAb of this invention may be cultivated in vitro orin vivo. Alternatively, techniques described for the production ofsingle chain antibodies can be adapted to produce differentiallyexpressed gene-single chain antibodies. Single chain antibodies areformed by linking the heavy and light chain fragments of the Fv regionvia an amino acid bridge, resulting in a single chain polypeptide.

Detection of the antibodies described herein may be achieved usingstandard ELISA, FACS analysis, and standard imaging techniques used invitro or in vivo. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, 3-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I ³⁵S or ³H.

For example, in a typical forward assay, unlabeled antibody isimmobilized on a solid substrate and the sample to be tested broughtinto contact with the bound molecule. After a suitable period ofincubation, for a period of time sufficient to allow formation of anantibody-antigen binary complex, a second antibody, labeled with areporter molecule capable of inducing a detectable signal, is added andincubated, allowing time sufficient for the formation of a ternarycomplex of antibody-antigen-labeled antibody. Any un-reacted material isthen washed away, and the presence of the antigen is determined byobservation of a signal, or may be quantified by comparing with acontrol sample containing known amounts of antigen. Variations on theforward assay include the simultaneous assay, in which both sample andantibody are added simultaneously to the bound antibody, or a reverseassay in which the labeled antibody and sample to be tested are firstcombined, incubated and added to the unlabeled surface bound antibody.These techniques are well known to those skilled in the art, and thepossibility of minor variations will be readily apparent. As usedherein, “sandwich assay” is intended to encompass all variations on thebasic two-site technique. For the immunoassays of the present invention,the only limiting factor is that the labeled antibody be an antibodywhich is specific for the p11 polypeptide or related regulatory protein,or fragments thereof.

The most commonly used reporter molecules are either enzymes,fluorophore- or radionuclide-containing molecules. In the case of anenzyme immunoassay an enzyme is conjugated to the second antibody,usually by means of glutaraldehyde or periodate. As will be readilyrecognized, however, a wide variety of different ligation techniquesexist, which are well-known to the skilled artisan. Commonly usedenzymes include horseradish peroxidase, glucose oxidase,beta-galactosidase and alkaline phosphatase, among others. Thesubstrates to be used with the specific enzymes are generally chosen forthe production, upon hydrolysis by the corresponding enzyme, of adetectable color change. For example, p-nitrophenyl phosphate issuitable for use with alkaline phosphatase conjugates; for peroxidaseconjugates, 1,2-phenylenediamine or toluidine are commonly used. It isalso possible to employ fluorogenic substrates, which yield afluorescent product rather than the chromogenic substrates noted above.A solution containing the appropriate substrate is then added to thetertiary complex. The substrate reacts with the enzyme linked to thesecond antibody, giving a qualitative visual signal, which may befurther quantitated, usually spectrophotometrically, to give anevaluation of the amount of polypeptide or polypeptide fragment ofinterest which is present in the serum sample.

Alternately, fluorescent compounds, such as fluorescein and rhodamine,may be chemically coupled to antibodies without altering their bindingcapacity. When activated by illumination with light of a particularwavelength, the fluorochrome-labeled antibody absorbs the light energy,inducing a state of excitability in the molecule, followed by emissionof the light at a characteristic longer wavelength. The emission appearsas a characteristic color visually detectable with a light microscope.Immunofluorescence and EIA techniques are both very well established inthe art and are particularly preferred for the present method. However,other reporter molecules, such as radioisotopes, chemiluminescent orbioluminescent molecules may also be employed. It will be readilyapparent to the skilled artisan how to vary the procedure to suit therequired use.

When the antibodies are intended for therapeutic use, it is preferredthat they have a human constant region so as to minimize theirimmunogenicity. Chimeric antibodies are made by splicing DNA encodingthe variable region from a donor antibody molecule of appropriateantigen specificity together with DNA encoding the constant region of ahuman antibody molecule. The antibodies may be further modified toprovide humanized antibodies, which are additionally modified to removenonhuman residues. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat, or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.The humanized antibody may comprise residues which are found neither inthe recipient antibody nor in the imported CDR or framework sequences.These modifications are made to further refine and optimize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region or domain (Fc), typically that of a humanimmunoglobulin. Alternatively, antibodies derived from nonhuman sourcesbut using fully human immunoglobulin genes may be made e.g., using phagedisplay techniques or transgenic animals, e.g., transgenic mice havinghuman IgV and IgC genes, and such antibodies should exhibit minimalimmunogenicity.

A “therapeutically effective amount” as used herein refers to an amountof drug sufficient to treat or ameliorate the pathological effects ofp11/5-HT receptor related disorders. For instance, a therapeuticallyeffective amount of p11 modulator sufficient to treat or ameliorate thepathological effects of a p11/5-HT receptor related disorder is anamount sufficient to either induce or inhibit p11 expression or regulate(either up or down) 5-HT_(1B) receptors levels at the neuronal plasmamembrane. Therefore, a therapeutically effective amount of p11 modulatorsufficient to treat or ameliorate the pathological effects of depressionis an amount sufficient to either induce p11 expression or increasep11's ability to recruit 5-HT_(1B) receptors to the neuronal plasmamembrane. Conversely, a therapeutically effective amount of p11modulator sufficient to treat or ameliorate the pathological effects ofanxiety disorders would be an amount sufficient to either inhibit p11expression or down-regulate 5-HT_(1B) receptors at the neuronal plasmamembrane. p11 modulator may be administered via known methods in the artincluding intravenous, subcutaneous, intramuscular, transdermal orintracerebral administration. Administration may be rapid as byinjection or over a period of time as by slow infusion or administrationof a slow release formulation.

The phrase “p11 knock-out” refers to a DNA sequence that has a total orpartial defect, alteration or mutation or is devoid or deficient in thep11 gene. A “p11 knock-out mouse” or “p11 knock-out transgenic mouse”therefore refers to a mouse wherein the DNA introduced into said mousecontains a defect, deficiency, mutation or alteration in the gene thatexpresses p11 proteins. As a result of the defect or deficiency in p11gene, a p11 knock-out mouse has fewer 5-HT_(1B) receptors at theneuronal plasma membrane and/or exhibits reduced or no 5-HT_(1B)receptors at the neuronal plasma membrane, thereby exhibiting adepression-like phenotype compared to wild-type mouse. The terms“knock-out” may refer to a deviation anywhere from 1 nucleotide todeletion of the entire gene compared to the original gene. Knock-outmice may be generated by using any known techniques in the art such astargeted homologous recombination.

The term “recombinant” refers to DNA which has been isolated from itsnative or endogenous source and modified either chemically orenzymatically to delete naturally-occurring flanking nucleotides orprovide flanking nucleotides that do not naturally occur. Flankingnucleotides are those nucleotides which are either upstream ordownstream from the described sequence or sub-sequence of nucleotides.

As used herein, a “vector” is something which delivers a recombinantnucleic acid to a desired cell or tissue, for example a virus which caninfect, transfect, or transiently or permanently transduce a cell. It isrecognized that a vector can be a naked nucleic acid, or a nucleic acidcomplexed with protein or lipid. The vector optionally comprises viralor bacterial nucleic acids and/or proteins, and/or membranes (e.g., acell membrane, a viral lipid envelope, etc.). For purposes of thisapplication, a vector may also be a cell comprising the recombinantnucleic acid. It is recognized that vectors typically include anexpression cassette placing the nucleic acid of interest under thecontrol of a promoter, or may simply include a promoter flanked bytargeting sequences to achieve insertion upstream of the gene whoseexpression is desired. Vectors include, but are not limited to replicons(e.g., plasmids, bacteriophages) to which fragments of DNA may beattached and become replicated. Vectors thus include, but are notlimited to RNA, autonomous self-replicating circular DNA (plasmids), andinclude both the expression and nonexpression plasmids. Where arecombinant microorganism or cell culture is described as hosting an“expression vector” this includes both extrachromosomal circular DNA andDNA that has been incorporated into the host chromosome(s). Where avector is being maintained by a host cell, the vector may either bestably replicated by the cells during mitosis as an autonomousstructure, or may be incorporated within the host's genome.

The phrase “nucleic acid sequence encoding” refers to a nucleic acidcontaining codons which, when transcribed and/or translated, express aspecific protein or peptide. The nucleic acid sequence may additionallycomprise flanking sequences, introns, and/or sequences encoding peptideswhich are subsequently cleaved post-translation. The nucleic acidsequences include both the DNA strand sequence that is transcribed intoRNA and the RNA sequence that is translated into protein. The nucleicacid sequences include both the full length nucleic acid sequences aswell as non-full length sequences derived from the full lengthsequences. It being further understood that the sequence includes thenative sequence as well as sequences utilizing degenerate codons, e.g.,to adapt the sequence to the codon preference in a specific host cell.

“Nucleic acids”, as used herein, may be DNA or RNA. Nucleic acids mayalso include modified nucleotides that permit correct read through by apolymerase and do not alter expression of a polypeptide encoded by thatnucleic acid.

EXAMPLES Example 1 Yeast Two-Hybrid Screen

To better understand the function of 5-HT_(1B) receptors, the thirdintracellular loop of this receptor is used as bait in a yeasttwo-hybrid screen. The third intracellular loop of the rat 5-HT1Breceptor (amino acids 226-311) is PCR amplified from a the full-lengthcDNA rat brain library and subcloned into the Nco I/Sal sites of a baitpAS2-derived vector, for expression as a GAL4 DNA-binding domain fusionprotein. The 5-HT_(1B) receptor-bait plasmid is transformed, by usingthe lithium acetate method, into yeast strain CG1945. The size andexpression level of the fusion protein are checked by immunoblot byusing an anti-GAL4 DNA binding domain antibody. The pACT2 rat brain cDNAlibrary is transformed into yeast strain Y187. Bait and preytransformants are mated on YPD medium and plated on medium (−LWH)selective for the expression of the histidine reporter gene. 244×106diploid clones from the pACT2 rat brain cDNA library are screened. Aftergrowth on this medium, a 5-bromo-4-chloro-3-indolyl-D-galactosideoverlay assay is performed. More than 300 clones grow on selectivemedium and are positive for the β-galactosidase reporter gene. Yeastextracts are prepared from double positive clones. Prey inserts areamplified by PCR (5′-CGCGTTTGGAATCACTACA GGGATG-3′ and5′-GAAATTGAGATGGTGCACGATGCAC-3′) and sequenced using a prey vectoroligonucleotide (5′-GGCTTACCCATACGATGTTC-3′). p11 is identified as themajor prey by a BLAST search. p11 prey plasmid clones are selectivelyrescued from the yeast, transformed into Escherichia coli for DNAamplification and retransformed into the yeast strain Y187 with (i) theoriginal 5-HT_(1B) receptor-bait vector, (ii) the bait control vector totest for transactivation, (iii) two other irrelevant bait constructs,pRP21 and CM 15, or (iv) bait constructs corresponding to the thirdintracellular loops of 5-HT1A, 5-HT2A, 5-HT5A, 5-HT6, D1 or D2receptors, to test the specificity of the interaction. The baitscorresponding to 5-HT_(1A) (amino acids 218-345), 5-HT_(2A) (amino acids236-302), 5-HT_(5A) (amino acids 233-295), 5-HT₆ (amino acids 209-265),D1 (amino acids 256-312) and D2 (amino acids 211-343) receptors,respectively, are made by PCR-amplification from the rat brain cDNAlibrary and subcloning into the pAS2-derived vector. Each of these baitsis co-transformed into yeast with the p11 prey construct and theinteraction analyzed by using −LW or −LWH medium and an X-gal overlayassay. All the co-transformants grew on the non-selective medium (−LW)control plates. On the selective medium (−LWH), a positive interactionof p11 with 5-HT1B receptors, but not with any of the other baits, isdetected.

Twenty-six out of 29 double positive prey clones encode the gene forp11. p11 interacts with 5-HT_(1B) receptors in this assay but not with5-HT_(1A), 5-HT_(2A), 5-HT_(5A), 5-HT₆, dopamine D₁ or dopamine D₂receptors, two irrelevant baits (Cdelta115 and pRP21), or the emptyplasmid, showing the specificity of p11's association with 5-HT_(1B)receptor.

Example 2 Co-Immunoprecipitation

HeLa cells, which contain endogenous p11 (S1), are grown in DMEM mediumto 60% confluence and transfected with pcDNA3.1-5-HT1BR-V5 or emptyplasmid constructs with Lipofectamine according to the manufacturer'sprotocol. After transfection, cell extracts are solubilized at 4° C. (in50 mM Tris, pH 7.4/150 mM NaCl/2 mM EDTA/2 mM EGTA/0.1% Triton andprotease inhibitors). Cell extracts are immunoprecipitated with anti-V5monoclonal antibody, incubated with protein G and thoroughly washed. Inother experiments, brain tissue from cerebral cortex of wild-type andp11 KO mice is homogenized in solubilization buffer at 4° C. Brainextracts are immunoprecipitated with a polyclonal 5-HT_(1B) receptorantibody, incubated with protein A and thoroughly washed. Theimmunoprecipitates froth the cells and the brain tissue are run out onan SDS-PAGE gel and transferred onto PVDF membranes. Immunoblotting iscarried out using a mouse monoclonal antibody against p11 (1/100).Antibody binding is detected by incubation with a secondary HRP-linkedantibody directed towards mouse IgG and enhanced chemiluminescence.

p11 coimmunoprecipitates with 5-HT_(1B) receptors in HeLa cells andbrain tissue.

Example 3 Immunofluorescence

HeLa cells are transfected with pcDNA3.1-5-HT1BR or pcDNA3.1-V5constructs. Thirty six hours posttransfection cells are fixed with 4%paraformaldehyde/0.01 M PBS for 10 min. Non-specific staining is blockedby incubation with 10% BSA in PBS. 5-HT_(1B) receptors and p11 arevisualized by incubation with anti-V5-FITC antibody (1/500) andanti-mouse p11 antibody (1/1000) followed by Alexa Fluor 568-labeledgoat anti-mouse secondary antibodies (1/500). After washing in PBS,cover slips are mounted on slides by using Gel/Mount. Images offluorescent proteins are acquired using a laser-scanning microscope.

Immunofluorescence shows a prominent colocalization between p11 and5-HT_(1B) receptors at the cell surface.

Example 4 In Situ Hybridization Experiments

All animal experiments are performed according to guidelines frominstitutional animal care committees at the Rockefeller University, theKarolinska Institute and the National Institutes of Health. Brains fromadult male Sprague Dawley rats are used to determine the regionaldistribution of p11 gene expression and its co-distribution with 5-HT1Breceptor gene expression. For some experiments, brains from p11 KO miceand their wild-type counterparts are used. To study the effects ofpsychoactive drug treatment on p11 mRNA expression, wild-type adult maleC57Bl6 mice are treated with a single injection or repeated injections(once daily for 14 days) of, vehicle, imipramine (10 mg/kg, i.p.),haloperidol (1 mg/kg, i.p.), diazepam (5 mg/kg, i.p), tranylcypromine(10 mg/kg, i.p.) or risperidone (1 mg/kg, i.p.). Animals are killed 1hour after the last injection. To study the effect of electroconvulsivetreatment (ECT) on p11 expression, male Sprague Dawley rats (200 gram)are exposed to daily ECT via ear clip electrodes (45 mA; 0.3 sec) for 10days and killed 18 hours after the last stimulation. Control animalsreceived sham treatment in which electrodes are clipped onto the ratears but no current is applied.

There is an upregulation of p11 mRNA in the forebrain following the 14day treatment with imipramine and with tranylcypromine, and followingthe repeated electroconvulsive therapy, but not with haloperidol,risperidone, or diazepam.

Example 5 p11 Protein Levels in Mouse Depression Model and in Normal andDepressed Humans

Wild-type adult male C57Bl6 mice are treated once daily for 14 days withvehicle or imipramine (10 mg/kg, i.p.) and killed 1 hour after the lastinjection. Male Sprague Dawley rats are exposed to daily ECT via earclip electrodes (45 mA; 0.3 sec) for 10 days and killed 18 hours afterthe last stimulation. Adult female helpless H/Rouen mice andnon-helpless NH/Rouen mice are sacrificed. From these 3 differenttreatment groups and their corresponding controls, frontal cortices aredissected out and frozen.

Fresh-frozen tissue from the human cingulate cortex of normal controlsand patients who suffered from major depression is obtained from theStanley Foundation Neuropathology Consortium. The frozen cortices aresonicated in 1% SDS and boiled for 10 min. Small aliquots of thehomogenate are retained for protein determination by the bicinchoninicacid protein assay method.

Equal amounts of protein are processed by using 10-20% gradientacrylamide gels. Immunoblotting is carried out with either polyclonal ormonoclonal antibodies against p11 (1/1000 for the human samples and1/200 for the rodent samples) and polyclonal antisera against actin(1/1000). Antibody binding is detected by enhanced chemiluminescence andquantified by densitometry, using National Institutes of Health IMAGE1.63 software. The level of p11 is normalized to the level of actin. Alldata are presented as normalized levels.

To study the regulation of p11 mRNA in a genetic mouse model ofdepression, forebrain tissue from adult female and male helpless H/Rouenmice and from non-helpless NH/Rouen mice are compared. p11 mRNA andprotein are markedly lower in H/Rouen mice. Similar results are foundwith the two genders.

Forty-am-thick cryostat-cut sections of human cingulated cortex ofnormal controls and patients who suffered from major depression areobtained from the Stanley Foundation Neuropathology Consortium. Theanalyzed samples are from both genders (6 females and 9 males in boththe normal and depression groups) and aged 29-68 (normal) and 30-65(depression) years. The duration of the disease among the depressedpatients varies from 1 to 42 years. Seven of the depressed individualsdied by suicide. The post-mortem intervals of the brain tissue before itis frozen are 8-42 (normal) and 7-47 (depression) hours and the pH ofthe tissue 5.8-6.6 (normal) and 5.9-6.5 (depression). In situhybridization probes are made by PCR amplification of nucleotides1159-1420 of the coding sequence of the rat 5-HT1B receptor gene,nucleotides 1-293 of the coding sequence of the mouse or human p11 genesand nucleotides 1-287 of the coding sequence of the rat p11 gene,respectively. The different PCR fragments are subcloned into thepCRII-TOPO vector. With the exception of the studies on human tissue,12-μm-thick cryostat sections are made for all studies. Sections arehybridized with [□⁻³⁵S]UTP-labeled riboprobe prepared by in vitrotranscription from cDNA corresponding to the rat 5-HT1B receptor gene ormouse, rat or human p11 gene as previously described (S5). Afterhybridization, the sections are exposed to Biomax MR film for 7 to 24days and analyzed using the NIH Image 1.63 software. Unless indicated,analyses are made in the prelimbic/anterior cingulate cortex. Somesections are dipped into Ilford K5 emulsion for cellular analysis. After8 weeks, the sections are developed, Nissl-stained and mounted.

Similar to the H/Rouen mice, p11 mRNA and protein are down-regulated inthe anterior cingulate cortex in patients who had suffered from unipolarmajor depression disorder.

Example 6 p11/5-HT_(1B) Receptor Co-Transfection Experiment

COS 7 cells, which contain low, if any, native p11, are transfected withp11 (pcDNA3.1-p11), with 5-HT_(1B) (pcDNA3.1-5-HT1BR-V5), with dopamineD₁ receptors (pcDNA3.1-D1R-V5), or empty plasmid. Plated COS 7 cells areincubated with medium containing 1 mg/ml Sulfo-NHS-LC-Biotin for 30 mMon ice. Cells are rinsed in TBS to quench the biotin reaction. Cells arelysed in 300 μl of modified RIPA buffer (1% Triton X-100, 0.1% SDS, 0.5%deoxycholic acid, 50 mM NaPO4, 150 mM NaCl, 2 mM EDTA, 50 mM NaF, 10 mMsodium pyrophosphate, 1 mM sodium orthovanadate, 1 mM PMSF, and 1 mg/mlleupeptin). The homogenates are centrifuged at 14,000 g for 15 min at 4°C. Fifteen μl of the supernatant are removed to measure total levels of5-HT_(1B) receptors. The remaining supernatant is incubated with 100 μlof 50% Neutravidin agarose for 3 hrs at 4° C. and briefly centrifuged.The supernatant, containing cytosolic 5-HT_(1B) receptors, is collected.Thereafter the agarose beads are washed 3 times with RIPA buffer and,after the final brief centrifugation, bound proteins are resuspended in40 μl of SDS sample buffer and boiled. Quantitative western blots areperformed on total, cytosolic and biotinylated (surface) proteins usinganti-V5 (to detect 5-HT_(1B) receptors; 1:1000) and anti-p11 (1:1000)antibodies. Immunoreactive bands are detected by enhancedchemiluminescence followed by autoradiography. The intensity of thebands is quantitated using NTH Image 1.63 software. The surface/totalratio is calculated for each well. Control experiments confirmed thatthe intracellular protein actin is not biotinylated in this assay.

Cells co-transfected with 5-HT_(1B) receptors and p11 exhibit more5-HT_(1B) at the cell surface than cells transfected with 5-HT_(1B)receptors alone. In contrast, the ratio of surface-to-total dopamine D₁receptors is similar in the presence of absence of p11.

Example 7 cAMP Measurements in COS 7 Cells

COS 7 cells grown in DMEM medium are transfected with 5-HT1B receptorsand/or p11. Thirty six hours later the cells are pretreated withtheophylline (5 mM) and pargyline (10 μM) for 15 min. Vehicle orforskolin (10 μM) with or without serotonin (10 μM), is then added foranother 15 minutes. At the end of the treatment, the drug-containingmedium is removed, the wells rinsed in PBS and the cells harvested. cAMPformation is quantitated by a direct cAMP enzyme immunoassay kitaccording to the manufacturers instructions. Control experiments showthat serotonin does not alter cAMP formation in untransfected COS 7cells.

The ability of serotonin (10 mM) to counteract forskolin-induced cAMPformation in COS-7 cells transfected with the 5-HT_(1B) receptor isincreased in the presence of cotransfected p11. There is no significantdifference in the cAMP responses to forskolin with or without p11. Dataare normalized to forskolin-stimulated conditions, with or without p11,and represent means T SEM.

Example 8 Generation and Analysis of Transgenic Mice Overexpressing p11

Transgenic mice with doxycycline-regulatable overexpression of p11 underthe calcium/calmodulin-dependent protein kinase II (CamKII) promoter aregenerated. Mouse p11 is fused with a Myc epitope tag using PCR andsubcloned into the Sal I/Hind III sites of pTet-splice (S6). Thisplasmid (pTetOp-p11-Myc) is transfected into tTA-expressing CHO cells(kind gift from Dr Patrick Allen). The expression of Myc from extractsof these cells is confirmed by immunoblotting using an anti-Myc (1:1000)antibody. After the confirmation that the p11-Myc is expressed, a DNAfragment (containing pTetOp-p11-Myc, SV40 intron, and poly(A)+signal) islinearized, purified by electroelution \and microinjected into thepronuclei of oocytes from C57BL6 mice and implanted in pseudopregnantC57BL6/CBA mice (Rockefeller University Transgenic Facility). Tail DNAis analyzed for the transgene by PCR (5′-TATAGTCGACATGATGCCATCCCAAATGG-3° and 5′-TATAAAGCTTCTAC AAATCTTCTTCAGAAATCAATTTTTGTTCAGATTTCTTCCCCTTCTG-3′). Founder mice positive for thepTetOp-p11-Myc construct are crossbred with C57Bl6 mice to generate F1mice. F2 homozygous pTetOp-p11-Myc-transgenic mice are obtained bycrossbreeding F1 siblings. (The homozygous genotype is confirmed bycrossbreeding them with wild-type mice.) These mice are crossbred withC57Bl6 mice expressing tTA under the CamKII promoter (S7). The mice aregenotyped by PCR with the abovementioned primers (to detect p11-Myc) and5′-GAGCTGCTTAATGAGGTCG GAATC-3′ and 5′-TCTAAAGGGCAAAAGTGAGTATGG-3′ (todetect tTA). The overexpression of p11-Myc in double transgenic mice isconfirmed by immunoblotting using an anti-Myc antibody and by in situhybrization against the mouse p11 gene (FIG. S2). The expression ofCamKII-driven tTA is detected using in situ hybrization with a riboprobeagainst the coding region of tTA (kind gift from Dr Alexei Morozov,Columbia University) (FIG. S2). In the behavioral experiments, doubletransgenic mice are compared with littermates expressing none or one thetransgenes serving as control mice. Some double transgenic mice receivedoxycycline 50 mg/l in their drinking water for 18 days before theexperiment.

In the absence of doxycycline, transgenic mice have elevated p11 inneurons that do not contain serotonin in the forebrain, but not inserotonin neurons in the raphe nuclei. These mice have increasedfunctional 5-HT_(1B) receptors in substantia nigra, and exhibit reducedthigmotaxis (an index of anxiety-related distress) and increasedhorizontal activity in the open-field test. They also show a decreasedimmobility in the tail suspension test (an index of depression-likestate). Thus, mice overexpressing p11 act as if they were treated withantidepressants, although a confounding factor is that they appear to begenerally hyperactive. Transgenic mice treated with doxycycline havenormalized p11 expression (FIG. S4) and no significant alterations ofthigmotaxis, immobility, or horizontal activities.

Example 9 Generation and Analysis of p11 Knock-Out Mice

Generation and Analysis of p11 ICO Mice.

Using a probe from the coding sequence of rat p11, 6 genomic clones areisolated from a BAC library screen. A 13.7-kb Bam HI fragment issubcloned from a BAC Clone and mapped by restriction enzyme analysis.The mouse p11 gene contains an ATG-containing exon, a 3.5-kb intron,followed by another exon with the stop codon. A 11.3 kb targeting vector(5′ Hinc II-Bgl II+[Bam H1-loxPNeoloxP-Kpn I]+Apa I*-Eco RV 3′) spanningthe ATG-containing exon of the p11 gene is made in pBSK(−) (FIG. S5).The targeting vector is electroporated into 129SvEv ES cells andselected for recombinant clones by G418. ES clones are identified aspositive for homologous recombination by southern blotting using 5° and3′ external probes (300-bp Bam HI/Bsp M1 fragment and 285-bp Bam HI/ScaI fragment, respectively) in an analysis of ES cell DNA digested withSpe I and Bam HI/Sal I, respectively. Positive clones are injected intoC57BL/6 blastocysts, and chimeric males are bred with C57BL/6 females toobtain germ-line transmission. Heterozygous offspring are mated togenerate knockout and wild-type mice. Southern blotting from tail DNAconfirmed that both alleles are mutated in p11 KO mice (FIG. S5). Theabsence of the p11 gene in the p11 knockout mice is further confirmedusing in situ hybridization with a probe against the mouse p11 gene(FIG. S5). A PCR procedure, using the following oligonucleotides,5′-CATTCAGAGGTGAACCCTGCTGAGGG-3′,5′-CCTGTCAGCCACTCTATAT GCTCCTAATC-3′and 5′-GGCCAGCTCATTCCTCCC ACTCATG-3′, is developed to distinguishwild-type, heterozygote and knockout mice (FIG. S5). This PCR-basedapproach is used for routine genotyping. Except for studies with primarycortical cultures, all experiments are performed on p11 KO and wild-typelittermates generated from heterozygote breeding. Theheterozygote×heterozygote breeding yielded 29% wild-type, 53% p11heterozygote and 18% p11 KO mice. It is unclear why there are fewer KOmice, but p11 has been found to be involved in early embryonicimplantation (S8). Heterozygote p11 mice are backcrossed for twogenerations with C57Bl6 mice. Microsatellite genotyping, using 104specific C57Bl6 markers (Rockefeller University Genomics ResourceCenter), shows that heterozygote p11 mice used for breeding ofexperimental animals are on a 74±2.8% C57Bl6 background.

Quantitative receptor autoradiography. Cryostat sections (12 μm thick)are made from p11 KO and wild-type mice. 5-HT_(1B) receptors aredetected by incubating the sections in 170 mM Tris/150 mM NaCl pH 7.4(25° C.) containing the antagonist [¹²⁵I]cyanopindolol (0.3, 1, 3, 10,30, 100 pM; 2200 Ci/mmol), 100 nM 8-OH-DPAT as a 5-HT_(1A) blocker, and30 μM isoproterenol, as β-adrenergic receptor blocker, for two hours(S9). Non-specific binding is determined by measurements in the presenceof 100 μM serotonin. In displacement experiments, increasingconcentrations of serotonin (0, 0.3, 1, 3, 10, 30, 100, 300, 1000, 10000nM) are incubated with 10 pM [¹²⁵I]cyanopindolol as described above.5-HT_(1B) receptors are also detected by incubating the sections in 9170 mM Tris/4 mM CaCl2/0.1% ascorbic acid pH 7.4 (25° C.) with theantagonist [³H]GR125743 (0.3, 1, 3, 10, 30 nM; 80 Ci/mmol; GEHealthcare) for two hours. Nonspecific binding is determined bymeasurements in the presence of 100 μM serotonin. 5-HT_(1A) receptorsare detected by incubating the sections in 50 mM Tris pH 7.4, 4 mMCaCl₂, 1 mM MgCl₂ and 0.1% bovine serum albumin (25° C.) with agonist[³H]8-hydroxy-2-(di-n-propylamino)-tetralin ([³H]8-OH-DPAT; 10 nM; 125Ci/mmol; GE Healthcare), 300 nM SB-269970, as a 5-HT7 receptor blocker,for one hour. Non-specific binding is determined by measurements in thepresence of 100 μM serotonin. D1-like receptors are detected byincubating the sections in 25 mM Tris/100 mM NaCl/1 mM MgCl₂/1 μMpargyline/20 nM mianserin/0.001% ascorbic acid containing the antagonist[³H]7-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepine-7-ol([³H]SCH 23390; 2 nM; 87.0 Ci/mmol) for 2 hours. Non-specific binding isdetermined by measurements in the presence of 100 μM SKF82958. D2-likereceptors are detected by incubating the sections with 170 mM Tris/120mM NaCl/5 mM KCl/2 mM CaCl₂,/1 mM MgCl₂,/10 μM GTP/0.001% ascorbic acidcontaining the antagonist [³H]raclopride (5 nM; 72 Ci/mmol) for onehour. Non-specific binding is determined by measurements in the presenceof 100 μM quinpirole. At the end of all autoradiographic experiments,sections are rinsed 2×5 min in their corresponding cold binding buffers,dipped in distilled water at 4° C. and dried under cold air. Thesections are apposed to Biomax MR films for 3-5 days([¹²⁵I]cyanopindolol) or 4-10 weeks ([³H]GR125743, [³H]8-OHDPAT,³H]SCH23390, [³H]raclopride) together with [¹²⁵I] or [³H] microscales.Optical density measurements are obtained in several brain regions withthe NIH Image 1.63 image analysis system. Specific binding is calculatedby digital subtraction of nonspecific labeling from total binding.Standard curves generated from [³H] or [¹²⁵I] microscales are used toconvert optical densities into femtomoles per milligram of protein. Dataobtained from saturation and displacement experiments are analyzed usingnon-linear regression equations.

Autoradiographic ligand-binding experiments showed that there are fewerbinding sites for the 5-HT_(1B) receptor antagonist radioligands[¹²⁵I]iodocyanopindolol and [³H]GR125743 in globus pallidus in p11 KOthan in wild-type mice. Similarly, [¹²⁵I]iodocyanopindolol binding islower in substantia nigra pars reticulata in p11 KO than in wild-typemice (77.3±5.8 versus 98.8±6.2 fmol/mg protein; P<0.05 Student's ttest). There is no difference in the affinity of serotonin to displacebound [¹²⁵I]iodocyanopindolol between wild-type and p11 KO mice [medianeffective concentration (EC₅₀) values: 57 versus 52 nM). No differencesin the amounts of 5-HT_(1A), D₁, or D₂ receptors are detected betweenthe wild-type and p11 KO mice. [¹²⁵I]Iodocyanopindolol binding is alsoreduced in H/Rouen mice versus NH/Rouen mice.

[³⁵S]GTPγS Binding in Response to 5-HT1A or 5-HT1B Receptor Stimulation.

Fresh cryostat sections (12 μm) from wild-type, p11 KO and p11transgenic mice are preincubated for 30 min in Tris-HCl 50 mM (pH 7.4)supplemented with 100 mM NaCl, \10 3 mM MgCl₂, 0.2 mM EGTA, 2 mM GDP and1 U/ml adenosine deaminase to remove endogenous adenosine. The sectionsare thereafter incubated for two hours at 25° C. in the same solutioncontaining 40 pM [³⁵S]GTP S, with (stimulation condition) or without(basal condition) 50 μM of the 5-HT1A receptor agonist, 8-OH-DPAT, orthe 5-HT1B receptor agonist, anpirtoline. Non-specific labeling(background) is determined on autoradiographs from adjacent sectionsincubated with 10 μM of unlabeled GTP S. Sections are rinsed twice (3min each) in 50 mM Tris-HCl buffer, once (30 sec) in distilled water toremove the buffer salts, and air-dried. The autoradiographs are obtainedby 2-4 days exposure on Biomax MR film. Optical density measurements areobtained in several brain regions with the NIH Image 1.63 image analysissystem.

The reduced number of 5-HT_(1B) receptors at the cell membrane in p11 KOmice is reflected in a reduced ability of the 5-HT_(1B) receptor agonistanpirtoline to increase [³⁵S]guanosine 5′-O-(3′-thiotriphosphate (GTP-S)binding in globus pallidus in these mice. In contrast, there is nodifference in [³⁵S]GTP-S binding by 8-OH-DPAT[(+/−)-8-hydroxy-2-(di-n-propylamino)tetralin], a 5-HT_(1A) receptoragonist, in wild-type and p11 KO mice (6.0±2.1 versus 5.0±2.0 opticaldensity units). The decreased number of functional 5-HT_(1B) receptorsat the cell surface of p11 KO mice is also reflected in a loss ofability of serotonin and of anpirtoline to down-regulatephospho-Thr²⁰²/Tyr²⁰⁴-ERK1/2 (extracellular signal-regulated kinase)levels in primary cortical cultures from p11 KO mice and of anpirtolineto decrease phospho-Ser⁹-synapsin I, a site phosphorylated bycAMP-dependent protein kinase, in striatal slices from p11 KO mice.

Western Blotting to Detect Phosphorylated ERK1/2 in Primary CorticalCultures.

Cortices are removed from E18 mice generated from WT×WT or p11KO×p11KObreedings, trypsinized (0.25%), dissociated by trituration and platedonto poly-L-lysine (1 mg/ml) coated six-well plates. The cultures(500,000 cells/ml) are grown in medium containing DMEM with 5% fetalbovine serum, 4 mM L-glutamine, B-27 nutrient supplement, penicillin (5U/ml), and streptomycin (5 μg/ml). After two weeks, the cultures aretreated with vehicle, serotonin (10 μM) or anpirtoline (10 μM) for 15minutes. At the end of the treatment, the drug-containing medium isremoved, the wells rinsed in ice-cold PBS, the neurons removed by a cellscraper and frozen in liquid nitrogen. Frozen cell samples are sonicatedin 1% SDS and boiled for 10 mM. Small aliquots of the homogenate areretained for protein determination by the bicinchoninic acid proteinassay method. Equal amounts of protein are processed by using 10%acrylamide gels, as described (S10). Immunoblotting is carried out witha phosphorylation-state-specific antibody againstphospho-Thr202/Tyr204-ERK1/2 or an antibody that is notphosphorylation-state-specific against total ERK1/2. Antibody binding isdetected by enhanced chemiluminescence and quantified by densitometry,using National Institutes of Health IMAGE 1.63 software. The level ofthe phosphorylated form of ERK1/2 is normalized to its total level. Alldata are presented as normalized levels.

Western Blotting to Detect Phosphorylated Synapsin I in Brain Slices.

Slices (300 μm) from the striatum are prepared from wild-type and p11 KOmice as described (S10). The slices are preincubated in Krebs buffer(118 mM NaCl/4.7 mM KCl/1.5 mM Mg2SO4/1.2 mM KH2PO4/25 mM NaHCO3/11.7 mMglucose/1.3 mM CaCl₂) at 30° C. under constant oxygenation (95% 02/5%CO2) for 60 min, with a change of buffer after 30 mM. Slices are treatedwith vehicle or anpirtoline (50 μM) for 2 mM. After drug treatment, thebuffer is removed, the slices are rapidly frozen on dry ice, sonicatedin 1% SDS and boiled for 10 min. Small aliquots of the homogenate areretained for protein determination by the bicinchoninic acid proteinassay method. Equal amounts of protein are processed by using 10%acrylamide gels. Immunoblotting is carried out with aphosphorylation-state-specific rabbit polyclonal antibody againstphospho-Ser9-synapsin I, a site that is phosphorylated by PKA andCamKII, or a rabbit polyclonal synapsin antibody that is notphosphorylation-state-specific. Antibody binding is detected by enhancedchemiluminescence and quantified by densitometry, using NationalInstitutes of Health IMAGE 1.63 software. The level of thephosphorylated form of synapsin is normalized to its total level. Alldata are presented as normalized levels.

Electrophysiology.

Male p11 KO and wild-type mice (4-7 weeks old) are decapitated underfluorothane anesthesia. Their brains are rapidly removed and coronalbrain slices (400 μm thick), containing the nucleus accumbens, areprepared with a microslicer. Slices are incubated, for at least 1 h, at32° C. in oxygenated (95% O2+5% CO2) artificial cerebrospinal fluid(aCSF) containing (in mM): 126 NaCl, 2.5 KCl, 1.2 NaH2PO4, 1.3 MgCl₂,2.4 CaCl₂, 10 glucose and 26 NaHCO3, pH 7.4. Slices are transferred to arecording chamber mounted on an upright microscope and are continuouslyperfused with oxygenated aCSF at 28° C. Extracellular field potentialsare recorded using a glass micropipette filled with aCSF positioned onthe slice surface in the nucleus accumbens. Signals are amplified 500times via an Axopatch 200B amplifier acquired at 10 kHz, filtered at 2kHz and recorded on a Computer using acquisition and pClamp9 dataanalysis software. Synaptic responses are evoked with a concentricbipolar stimulating electrode placed near the recording electrode on thesurface of the slice. The dependence of the intensity of the stimulationapplied to the slice on the fEPSP amplitude is similar in WT and p11 KOmice, demonstrating that glutamatergic 12 synaptic transmission did notdiffer between WT and p11 KO mice. Single stimuli (0.1 ms duration) areapplied every 15 sec at an intensity yielding 50-70% maximal response asassessed by a stimulus/response curve established for each sliceexamined by measuring the amplitude of the field potential evoked byincreasing stimulus intensities. To evaluate the effect of serotoninreceptor activation on glutamatergic synaptic transmission, serotonin isapplied in the perfusion solution while measuring fEPSP/PS amplitude.Serotonin (50 μM; in the presence of 10 μM fluoxetine) depresses theamplitude of the fEPSP/PS in slices from WT mice (72 □□2.7% of baselinevalue) and this effect is abolished in slices from p11 KO mice (98□□3.6% of baseline value). Numerical values are expressed as means□□SEM. Drugs are applied in the perfusion solution by switching athree-way tap.

Serotonin, via 5-HT_(1B) receptors, reduces glutamate release atterminals of neurons originating from the cerebral cortex and inhibitssynaptic transmission at corticostriatal synapses. The amplitude offield excitatory postsynaptic potentials (f EPSPs) evoked by briefelectrical stimulation of glutamatergic fibers and recordedextracellularly in the nucleus accumbens is monitored. f EPSPs aremediated by AMPA receptors activated by endogenous glutamate released byelectrical stimulation of the slice in both wild-type and p11 KO mice [fEPSP/population spike (PS) reduction 77 and 81%, respectively, comparedwith baseline, 15 min after the AMPA receptor antagonist6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)]. When applied in theperfusion solution, serotonin depresses the amplitude of the f EPSP/PSin slices from wild-type mice, but not from p11 KO mice.

Tissue Content of Monoamines and Metabolites.

Male p11 KO, p11 heterozygote and wild-type mice (n=8 per genotype) aresacrificed by focused microwave irradiation and striata, cortices andhippocampi dissected out and frozen on dry ice. The tissue samples arethen sonicated in 10 volumes of 0.1N TCA, vortexed and centrifuged at12,000 g for 2 min. Supernatants are collected and analyzed forserotonin and the serotonin metabolite 5-hydroxyindolacetic acid(5-HIAA) using HPLC coupled with electrochemical detection (HPLC-EC).Serotonin and 5-HIAA are separated with a base deactivatedsilica-Hypersil 5 μm C18 analytical column (4.6×150 mm) with a mobilephase consisting of 75 mM sodium phosphate monobasic, 350 mg/L1-octanesulfonic acid sodium salt, 0.5 mM EDTA, 0.8% tetrahydrofuran(HPLC grade, inhibitor-free), and 8% acetonitrile, pH 3 (adjusted withphosphoric acid), at a flow rate of 1.2 ml/min. An electrochemicaldetector with dual glassy carbon electrodes is used (electrode 1=680 mV,range, 0.5 nA; electrode 2=−100 mV, range, 0.2 nA). Data are collectedusing EZChrom software that calculated peak heights and sampleconcentrations. The sensitivity for serotonin and 5-HIAA is 0.1 pmol/ml.

5-HT_(1B) receptors act as autoreceptors and inhibit serotonin release.Because p11 is expressed in the raphe nuclei, the amounts of serotoninand its major metabolite 5-hydroxyindoleacetic acid (5-HIAA) aremeasured in projection areas, namely, cortex, striatum, and hippocampusin wild-type and p11 KO mice. In accordance with a negative regulationof 5-HT turnover and/or metabolism by 5-HT_(1B) receptors, and apotentiating role of p11 on 5-HT_(1B) receptor function, p11 KO micehave increased levels of serotonin turnover and/or metabolism.

Behavioral Analyses—Open-Field Analysis.

Horizontal activities are measured for 30 minutes (results are analyzedevery 5 min period) during daytime in a fully computerized, multicage,red and infrared-sensitive motion detection system. The peripheralactivity values are divided 13 by the total horizontal activity valuesto determine thigmotaxis. In experiments with anpirtoline (5 mg/kg,i.p.) animals are tested 15 min postinjection. Some anpirtolinetreatedmice had received daily injections with imipramine (10 mg/kg, i.p.) for4 weeks until the day before the experiment.

Behavioral Analyses—Tail Suspension Test.

The tail suspension test, a model of antidepressant-like activity, iscarried out as described (S11) and is a modified version of thatvalidated for C57Bl6 (S12) and NMRI mice (S13). Mice are individuallysuspended by the tail to a horizontal bar (distance from floor is 35 cm)using adhesive tape (distance from tip of tail is 2 cm). Typically, micedemonstrated several escape-oriented behaviors interspersed withtemporally increasing bouts of immobility. A 6-min test session isvideotaped and scored by an observer who is unaware of the genotype. Theparameter recorded is the number of seconds spent immobile. Inexperiments with anpirtoline (5 mg/kg) and imipramine (10 mg/kg),animals are tested 15 minutes postinjection.

Behavioral Analyses—Sucrose Consumption Test.

A single bottle procedure in individually housed p11 KO mice andwild-type mice is used for testing sucrose consumption. Consumption of a2% sucrose solution in water is measured during a 96-hour period. In asubsequent experiment, water intake is measured for the same period oftime.

To evaluate behavioral effects of p11 deletion, thigmotaxis in wild-typeand p11 KO mice is compared under basal conditions and in response toanpirtoline in drug-naïve mice and in mice that have been treatedlong-term with imipramine. In animals treated with imipramine,anpirtoline causes a significant reduction in thigmotaxis in wild-typemice, but not in p11 KO mice (FIG. 4G). In addition, there is lessthigmotaxis in saline-injected wild-type than p11 KO mice (FIG. 4G).Drug-naïve wild-type and p11 KO mice exhibit similar thigmotaxis eitherin the absence or presence of anpirtoline. There is an increasedimmobility in the tail suspension test in p11 KO mice compared withwild-type mice, both under baseline conditions and after acute treatmentwith either anpirtoline or imipramine (FIG. 4H). These behavioralresults indicate that p11 KO mice exhibit a depression-like phenotypeand that p11 mediates behavioral responses to imipramine via 5-HT_(1B)receptors. In further support of a depression-like phenotype of p11 KOmice, p11 KO mice consume less of a palatable 2% sucrose solution thantheir wild-type littermates (1.74±0.07 versus 2.17±0.11 ml/g body weightper day; P<0.05 Student's t test), which indicates a decreasedresponsiveness to sweet reward. Water intake is similar in the p11 KOmice and their wild-type littermates (1.51±0.05 versus 1.42±0.05 ml/gbody weight per day), which rules out a role of altered fluid balance inthis behavior.

Example 10 Detection of p11 in Human Peripheral Blood Mononuclear Cells(PBMC)

Whole blood(10-15 ml) is collected in a heparinized tube. One ml ofblood will yield approximately 1 milj mononuclear cells, although thenumber varies considerably between individuals. The blood is diluted 1:1in phosphate-based saline (PBS). 2.5 ml lymphoprep (Medinor cat nr1114547) is added to 15 ml tubes and 10 ml diluted blood carefullylayered on top. The tubes are spun 20 minutes at 1800 rpm and roomtemperature. Using a Pasteur pipette, PBMC are collected from two tubesinto one clean 15 ml tube. This tube is filled with PBS and spun down at1500 rpm for 10 minutes. The cells are then washed twice in PBS (removesblood platelets as well as lymphoprep). To count, PBMC are diluted in 1ml of medium (90% fetal calf serum/10% DMSO) per original 10 ml wholeblood volume. (Count diluting 1:10 in Trypan Blue). PBMC are frozen at−80° C. (dry ice) or Cooler, and stored it and delivered at −80° C. 0.5milj PBMC per well are added to 96-well plates. The plate is spun andthe supernatant discarded. The cells are fixed in BD fixation buffer (BDBiosciences, from kit cat no 554715). The permeabilisation buffer fromthe same kit is added and the cells are washed. The cells are now readyfor intracellular staining. p11 antibody (BD Biosciences; 2.5 ug/ml) isdiluted in the permeabilisation buffer and added to the wells. One wellis used for IgG1 control. The cells are suspended by pipetting. Thecells are incubated for 30 minutes and washed in permeabilizationbuffer. The secondary antibody (ex goat anti-mouse PE conjugated) isdiluted in permeabilization buffer and added to each well. The cells aresuspended and incubated for 30 minutes. They are washed twice withpermeabilization buffer and once with PBS-1% FCS. For double-staining,the secondary antibody is blocked by washing once with 100 ul PBS-1%FCS-1% NMS (normal mouse serum). The blocking step is necessary to avoidbinding of subsequent antibodies to any remaining secondary antibody. Tostain surface markers, PBS containing CD14-PerCP (to distinguishmonocytes) or CD3-PerCP and CD56-PE (to distinguish T cells and NKcells), or CD19-FITC (to distinguish B cells) is added to each well. Thecells are suspended by pipetting and incubated 10 minutes in therefrigerator. They are then washed once with 200 ul PBS-1% FCS. StandardFACS procedure is used to determine the staining of p11 in the differenttypes of mononuclear cells. p11 is highly expressed in some white bloodcells, monocytes, NK killer cells and CD-8 positive T-cells.

1-39. (canceled)
 40. A method of diagnosing a p11/5-HT receptor relateddisorders in a subject comprising determining the level of p11 in abiological sample wherein said sample comprises peripheral bloodmononuclear cells from said subject and comparing said p11 level to areference, wherein an abnormal level of p11 compared to the referenceconstitutes a positive diagnosis of p11/5-HT receptor related disorder.41. The method according to claim 40, wherein said peripheral bloodmononuclear cells are NK cells and/or CD-8+ T-cells.
 42. The methodaccording to claim 40, wherein said peripheral blood mononuclear cellsare NK cells.
 43. The method according to claim 40, wherein saidp11/5-HT receptor related disorder is depression.
 44. The methodaccording to claim 43, wherein said depression comprises a p11associated depression.
 45. The method according to claim 40, whereinsaid p11/5-HT receptor related disorder is a disorder associated with anabnormally low level of p11, wherein reduced level of p11 in a subjectcompared to a control subject constitutes a positive diagnosis of suchdisorders.
 46. The method according to claim 45, wherein said disorderassociated with an abnormally low level of p11 is selected from a groupconsisting of depression, obsessive compulsive disorder, drug addiction,eating disorders, Alzheimer's disease, attention deficit disorder andattention deficit hyperactive disorder.
 47. The method according toclaim 40, wherein said p11/5-HT receptor related disorders are disordersassociated with an abnormally high level of p11, wherein an elevatedlevel of p11 in a subject compared to that in a control subjectconstitutes a positive diagnosis of such disorders.
 48. The methodaccording to claim 47, wherein said disorder associated with anabnormally high level of p11 is selected from a group consisting ofmania, dipolar disorder, anxiety disorders, aggression, sleep disorders,sexual dysfunction and gastrointestinal disorders.
 49. The methodaccording to claim 40, wherein said level of p11 is determined byassaying p11 protein level in the monocytes and/or lymphocytes in saidbiological sample from said subject.
 50. The method according to claim40, wherein said level of p11 is determined by assaying p11 mRNA levelin a biological sample from said subject.
 51. The method of claim 40,wherein the level of p11 is determined using a monoclonal antibodyspecific for p11.
 52. The method of claim 50, wherein the detection ofsaid monoclonal antibody is achieved by using standard ELISA, FACSanalysis, and/or standard imaging techniques.